Halomonas as a chassis

Abstract With the rapid development of systems and synthetic biology, the non-model bacteria, Halomonas spp., have been developed recently to become a cost-competitive platform for producing a variety of products including polyesters, chemicals and proteins owing to their contamination resistance and ability of high cell density growth at alkaline pH and high salt concentration. These salt-loving microbes can partially solve the challenges of current industrial biotechnology (CIB) which requires high energy-consuming sterilization to prevent contamination as CIB is based on traditional chassis, typically, Escherichia coli, Bacillus subtilis, Pseudomonas putida and Corynebacterium glutamicum. The advantages and current status of Halomonas spp. including their molecular biology and metabolic engineering approaches as well as their applications are reviewed here. Moreover, a systematic strain engineering streamline, including product-based host development, genetic parts mining, static and dynamic optimization of modularized pathways and bioprocess-inspired cell engineering are summarized. All of these developments result in the term called next-generation industrial biotechnology (NGIB). Increasing efforts are made to develop their versatile cell factories powered by synthetic biology to demonstrate a new biomanufacturing strategy under open and continuous processes with significant cost-reduction on process complexity, energy, substrates and fresh water consumption.


Introduction
Due to the sustainable and environment-friendly advantages of industrial biotechnology [1-4], it has been developed for decades to produce numerous bio-based products such as bioplastics [5][6][7], medicines [8], chemicals, food additives [9,10] and biofuels [11][12][13], with an aim to gradually replace the traditional petrol-based industry. However, the long-standing challenges of contamination risk, high sterile energy input and high fresh water consumption involved in bioprocesses of current industrial biotechnology (CIB) still reduce its competitiveness in spite of the rapid development of synthetic biology [14,15].
To make industrial biotechnology competitive to chemical industry, suitable microorganisms should be constructed to solve the existing difficulties of CIB [16]. Recently, the salt-tolerating bacteria, Halomonas spp., are becoming the attractive candidate hosts for microbial cell factory engineering due to their strong metabolism of diverse substrates and fast growth under high salt and high pH conditions, making possible the contamination free, non-food raw materials-and seawater-consuming fermentation processes [17,18]. In contrast with the traditional model microorganisms, such as Escherichia coli [14,19], Bacillus subtilis [20], Pseudomonas putida [21] and others [22], the development of molecular manipulation tools and methods for Halomonas spp. is difficult and less effective [23]. Recent attempts have been successful to establish genetic engineering approaches [24,25] including genome editing and gene expression control [26] to construct Halomonas spp. into a diverse bioproduct producers.
It is important to note that, Halomonas bluephagenesis, one of the well-studied salt-loving wildtype microbe able to accumulate poly-3-hydroxybutyrate (PHB), has been reprogrammed to be a low-cost chassis for various biosyntheses conducted under seawater-based unsterile open fermentation [27]. This Halomonas-based biotechnology, namely, the 'next-generation industrial biotechnology' (NGIB), has

Genetic manipulations in Halomonas spp.
Due to the recent growing interest in Halomonas spp. used as microbial chassis, many efforts have been made to develop genetic tools, such as expression vectors [59,60], promoters [24,61], ribosome-binding sites (RBSs), inducible systems [25], genome editing tools [26] and pathway tuning approaches [42,62,63], allowing genetic manipulations of Halomonas spp. possible for strain improvements [64]. For a given module of gene over-expression in model microbes such as E. coli and P. putida, several parts are necessary including vectors containing relative antibiotic-resistant gene(s) and replicon, promoters, RBSs, coding sequences and terminators [65]. Insulators are sometimes needed to minimize the unwanted sequence context between two different gene parts [66]. Generally, transformation of broad host range genetic parts into Halomonas spp. is an efficient strategy to screen suitable gene expression elements [23]. However, native plasmids isolated from Gram-negative halophiles are also good choice for cloning uses [67]. Till now, many expression vectors including pSEVA plasmids [68], shuttle vectors pWL102 and pUBP2 [69] and other broad host range or native plasmids are found to be usable in Halomonas spp. Accordingly, antibiotics such as chloromycetin (Cm) and spectinomycin (Spe) have been proven effective for Halomonas spp [70]. More importantly, a porin constitutive promoter library consisting of a wide range of transcriptional strengths, a novel type of T7-like inducible system and expression variances cross different expression systems were developed and characterized in Halomonas spp. for gene expression tuning [25,61,63] ( Figure 3A). Moreover, recent efforts on whole-genome sequencing (WGS) of over 450 Halomonas spp. strains (Supplementary Table S1), including H. bluephagenesis (also termed Halomonas TD01), Halomonas smyrnensis AAD6 [71,72], Halomonas sp. KM-1 [73], Halomonas sp. HAL1 [74] and so on, provides plentiful genomic information for boosting the developments of endogenous genetic parts mining, valuable pathways identification, metabolic networks modeling [72,75] etc., which offers fundamental insights into rational microbial cell factory engineering based on Halomonas sp. [35,[75][76][77]. Genome editing tools including homologous recombination and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 have been commonly used for site-specific mutagenesis in many microorganisms [78]. However, there were less studies reported in Halomonas spp. [26,79] except some successful cases based on double cross-overs homologous recombination using lethal genes or helper plasmids as selection pressures. An essential gene-deficient mutant can be used as the host (in the presence of the gene encoded in a plasmid), for example, the deletion of gene pryF encoding orotidine-5 -phosphate decarboxylase, can significantly improve the selection pressure during mutagenesis, especially for essential gene deletion and large deoxyribonucleic acid (DNA) fragment integration [80,81]. Recently, a CRISPR/Cas9-based gene editing tool was established in H. bluephagenesis in the authors' lab for engineering chromosomes, such as gene knock-down for morphology control [80], bypass deletion for product flux enhancement [82], target module integration on to the chromosome [63]. All of these efforts help turn Halomonas spp. into a reprogrammable chassis comparable with other model microbes ( Figure 1).

Figure 2. Metabolic pathways for diverse bioproductions by engineered Halomonas
Halomonas spp. have been engineered to produce different chemicals and polyesters from glucose, fatty acids, gluconate or other structure-related carbon sources (light gray). Three major metabolic modules involved in glucose metabolism, PHA biosynthesis (light blue), and productions of amino acids and their derivates (light orange) are summarized according to the published studies.
Crosses in red represent bypasses (letters in light gray) required to be knockout. Currently, conjugation is still the most commonly used genetic transformation method for Halomonas spp. due to the unsolved difficulty of electroporation or chemical transformation of expression vectors [79]. Therefore, many high throughput-dependent methods are not useful for Halomonas spp. engineering, resulting in challenges of attempts in large dataset mining and analysis when leveraging 'Design-Build-Test-Learn' cycle [83].

Metabolic engineering in Halomonas spp.
Metabolic engineering generally requires exquisite expression tuning of target pathways to channel the metabolic flux towards metabolic targets leveraging different static optimization approaches in addition to the commonly used methods, including enzyme engineering, promoter engineering, RBS optimization, gene over-expression (on/off-control only) and bypass knockout (Figure 2). Recently, an approach termed high-resolution gene expression control was developed for H. bluephagenesis based on the combination of two isopropyl-β-d-thiogalactopyranoside (IPTG)-induced systems with different dynamic ranges [63]. Similar to the length measuring tools with different scales of measuring ranges, this approach allows precise transcription tuning of target genes on chromosome directly in cross-magnitude scopes in corporation of green fluorescent protein (GFP)-mediated transcriptional mapping strategy [63]. On the basis of these successes, two orthogonal inducible systems induced by acyl homoserine lactone (AHL) and IPTG, respectively, were used to fine-tune two individual expression modules simultaneously, resulting in over 12-folds improvement of ectoine titer by engineered H. bluephagenesis [42]. This attempt gives a successful example for high throughput-independent strain engineering paradigm without large size library construction and labor-intensive screening process ( Figure 3B), which are helpful for non-model bacterial engineering with low efficient transformation methods, such as conjugation.
In addition to the metabolic engineering approaches, several strategies have been employed to achieve strain improvements for Halomonas spp.: (I) morphology engineering to enlarge cell shapes for enhanced intracellular substances (such as PHA or proteins) accumulation and for benefiting sedimentation during the separation process [80]; (II) introduction of bacterial hemoglobin, Vitreoscilla hemoglobin (VHb) for enhanced oxygen uptake leading to higher cell mass and more product formation as well as aeration energy saving [88]; (III) self-flocculation for convenient separation and wastewater-less bioprocessing by recycling the supernatant of fermented broth after heat treatment and membrane processing with a recycling rate of 70-85% [89]; (IV) control of redox potential nicotinamide adenine dinucleotide plus hydrogen (NADH)/nicotinamide adenine dinucleotide plus (NAD + ) by supplementing acetate to improve PHA and biomass accumulation ( Figure 3C) [90]. All of these strategies extend ability of Halomonas spp.
Halomonas spp. have been successfully engineered in previous studies including cell engineering, pathway optimization and process scale-up ( Figure 3D). Firstly, DNA sequence-based fundamental understanding of the selected Halomonas sp. and the genetic tools mining thereof is the cornerstone for strain engineering. Generally, the genome-wide sequencing and annotation can provide us a predictable overview of functional gene sets and metabolic networks [91]. Growth and characterization are important to obtain cultural protocols for various engineered purposes in different scales, including 96 deep well-plates, shake flasks and bench-top bioreactors, their results serve as reference-standards for growth profiling and pre-knowledge of recombinant cells without performing the scale-up studies [63], these are especially important as Halomonas spp. have NaCl concentrations and pH preferences.
Most importantly, optimization of genetic parts, promoter, RBS [49] and tools, promoter library construction [92], inducible systems [93], effective CRISPR-based gene editing approach [94] and alternative expression vectors, have been demonstrated to strongly improve the programmability of Halomonas. Screening on chromosomal locus with low expression variance can provide applicable genomic sites for efficient integrations of multiple expression modules [95]. Secondly, assembling catalytic enzymes to rewire the endogenous flux towards metabolic targets is commonly employed to achieve prototype success of target products from 0 to 1, namely pathway construction. Further optimization leveraging static regulation, mainly refers to bypass deletion and flux tuning, and dynamic regulation [96] allowing gene expression control over time and levels, is a proven strategy to generate significant breakthrough from 1 to 100. Thirdly, a rigorous scale-up test including medium and feeding strategy optimization can obtain iterative bioprocess of refinement. Accordingly, many fundamental insights can also be uncovered during the growth assays in corporation of omics profiling [97] and adaptive evolution [98], directing the redesign of cell factory by implementing 'Design-Build-Test' cycle, this is also true for Halomonas spp.
Many bioprocess-inspired strategies are useful in real cases, such as morphology control and self-flocculating cells aiming to make continuous fermentation and easier downstream separation possible. For compiling these three parts into a closed loop streamline, the interactive relationship among 'host engineering' , 'downstream engineering' and 'scale-up' is usually missed ( Figure 3D), however, they should be combined into strain engineering so that the engineering concept is used throughout the production. The NGIB concept based on extremophilic bacteria proposed by the authors' lab has demonstrated the engineering concept up-and down-stream of the bioproduction [64]. Recent efforts have also exemplified a successful case in rebuilding the yeast system for obtaining a metabolic target-dependent (or -preferred) host to realize enhanced production of fatty acids before the execution of pathway optimization [98].

Opportunities and challenges
Compared with the well-studied chassis especially E. coli and C. glutamicum, Halomonas spp. displaying several advantages including contamination-resistance, fast growth, seawater-based media and wide range substrates utilization, added with rich engineering tools and approaches developed recently, have become a promising platform for NGIB. A variety of products have been successfully produced with some of them scaled up to at least 5000 liters bioreactors under open unsterile conditions. Since Halomonas spp. can be engineered as convenient as E. coli, their potentials will be at least as promising as E. coli which has been employed to produce many bio-based products in various scales. Halomonas spp.-based NGIB has provided a versatile low-cost platform of biomanufacturing to meet the increasing demand of sustainable development that chemical industry and CIB cannot realize.
More efforts and attempts should be made to strengthen the sciences and technological sites of NGIB for overcoming the accompanying challenges. For example, the development of high throughput genetic transformation methods to enable generation of large size datasets from clones [49], establishments of multigene pathway tuning strategy [93] and dynamic control systems [99], exploration of high cell density cultivation and induction technology [100], enhancement of substrates to product conversion efficiency, controllable cell morphology changes for better growth, production and downstream processing. The coming joint efforts offer more possibilities for Halomonas as a chassis comparable with E. coli yet with more advantages.

Summary
• Halomonas spp. as chassis are able to grow rapidly in saline and alkaline environments allowing contamination-free cultivation under open conditions.
• Halomonas spp. display advantages including fast growth, seawater-based medium and a wide substrate range as well as simple cultivation processes.
• Similar to E. coli, Halomonas spp. can be engineered to produce diverse products including several PHAs, chemicals, food additives and proteins.
• H. bluephagenesis is one of the most promising hosts as it allows convenient genetic manipulations and process scale-up under open unsterile conditions.
• NGIB based on Halomonas spp. as chassis provides a competitive solution for overcoming the challenges of high energy and fresh water consumption based on CIB.

Author Contribution
J.-W.Y. and G.-Q.C. wrote the paper and drafted the figures.