Adapting enzymes to improve their functionality in plants: why and how.

Synthetic biology creates new metabolic processes and improves existing ones using engineered or natural enzymes. These enzymes are often sourced from cells that differ from those in the target plant organ with respect to, e.g. redox potential, effector levels, or proteostasis machinery. Non-native enzymes may thus need to be adapted to work well in their new plant context ('plantized') even if their specificity and kinetics in vitro are adequate. Hence there are two distinct ways in which an enzyme destined for use in plants can require improvement: In catalytic properties such as substrate and product specificity, kcat, and KM; and in general compatibility with the milieu of cells that express the enzyme. Continuous directed evolution systems can deliver both types of improvement and are so far the most broadly effective way to deliver the second type. Accordingly, in this review we provide a short account of continuous evolution methods, emphasizing the yeast OrthoRep system because of its suitability for plant applications. We then cover the down-to-earth and increasingly urgent issues of which enzymes and enzyme properties can - or cannot - be improved in theory, and which in practice are the best to target for crop improvement, i.e. those that are realistically improvable and important enough to warrant deploying continuous directed evolution. We take horticultural crops as examples because of the opportunities they present and to sharpen the focus.

Running the numbers on plant synthetic biology solutions to global problems.

Synthetic biology and metabolic engineering promise to deliver sustainable solutions to global problems such as phasing out fossil fuels and replacing industrial nitrogen fixation. While this promise is real, scale matters, and so do knock-on effects of implementing solutions. Both scale and knock-on effects can be estimated by 'Fermi calculations' (aka 'back-of-envelope calculations') that use uncontroversial input data plus simple arithmetic to reach rough but reliable conclusions. Here, we illustrate how this is done and how informative it can be using two cases: oilcane (sugarcane engineered to accumulate triglycerides instead of sugar) as a source of bio-jet fuel, and nitrogen fixation by bacteria in mucilage secreted by maize aerial roots. We estimate that oilcane could meet no more than about 1% of today's U.S. jet fuel demand if grown on all current U.S. sugarcane land and that, if cane land were expanded to meet two-thirds of this demand, the fertilizer and refinery requirements would create a large carbon footprint. Conversely, we estimate that nitrogen fixation in aerial-root mucilage could replace up to 10% of the fertilizer nitrogen applied to U.S. maize, that 2% of plant carbon income used for growth would suffice to fuel the fixation, and that this extra carbon consumption would likely reduce grain yield only slightly.

Directed Evolution of Aerotolerance in Sulfide-Dependent Thiazole Synthases.

Sulfide-dependent THI4 thiazole synthases could potentially be used to replace plant cysteine-dependent suicide THI4s, whose high protein turnover rates make thiamin synthesis exceptionally energy-expensive. However, sulfide-dependent THI4s are anaerobic or microoxic enzymes and hence unadapted to the aerobic conditions in plants; they are also slow enzymes (kcat < 1 h-1). To improve aerotolerance and activity, we applied continuous directed evolution under aerobic conditions in the yeast OrthoRep system to two sulfide-dependent bacterial THI4s. Seven beneficial single mutations were identified, of which five lie in the active-site cleft predicted by structural modeling and two recapitulate features of naturally aerotolerant THI4s. That single mutations gave substantial improvements suggests that further advance under selection will be possible by stacking mutations. This proof-of-concept study established that the performance of sulfide-dependent THI4s in aerobic conditions is evolvable and, more generally, that yeast OrthoRep provides a plant-like bridge to adapt nonplant enzymes to work better in plants.

Engineering Burkholderia sacchari to enhance poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] production from xylose and hexanoate.

Burkholderia sacchari LFM101 LMG19450T is a Brazilian bacterium isolated from sugarcane crops soil and a promising biotechnological platform for bioprocesses. It is an efficient producer of poly(3-hydroxybutyrate) from carbohydrates including xylose. In the present work, the expression of B. sacchari xylose consumption genes (xylA, xylB and tktA) was combined with the expression of Aeromonas sp. phaC (PHA synthase), aiming to increase both the growth rates in xylose and the 3-hydroxyhexanoate (3HHx) molar fractions in the produced PHA. Genes were cloned into pBBR1MCS-2 vectors and then expressed in the B. sacchari PHA- mutant LFM344. Maximum specific growth rates on xylose and PHA accumulation capacity of all recombinants were evaluated. In bioreactor experiments, up to 55.5 % CDW was accumulated as copolymer, hexanoate conversion to 3HHx raised from 2 % to 54 % of the maximum theoretical value, compared to wild type. 3HHx mol% ranged from 8 to 35, and molecular weights were between 111 and 220 kg/mol. Thermal analysis measurement showed a decrease in Tg and Tm values with higher 3HHx fraction, indicating improved thermomechanical characteristics. Recombinants construction and bioreactor strategies allowed the production of P(3HB-co-3HHx) with controlled monomeric composition from xylose and hexanoate, allowing its application in diverse fields, including the medical area.

Investigating Nutrient Limitation Role on Improvement of Growth and Poly(3-Hydroxybutyrate) Accumulation by Burkholderia sacchari LMG 19450 From Xylose as the Sole Carbon Source.

Burkholderia sacchari LMG19450, a non-model organism and a promising microbial platform, was studied to determine nutrient limitation impact on poly(3-hydroxybutyrate) [P(3HB)] production and bacterial growth from xylose, a major hemicellulosic residue. Nitrogen and phosphorus limitations have been studied in a number of cases to enhance PHA accumulation, but not combining xylose and B. sacchari. Within this strategy, it was sought to understand how to control PHA production and even modulate monomer composition. Nitrogen-limited and phosphorus-limited fed-batch experiments in bioreactors were performed to evaluate each one's influence on cell growth and poly(3-hydroxybutyrate) production. The mineral medium composition was defined based on yields calculated from typical results so that nitrogen was available during phosphorus limitation and residual phosphorus was available when limiting nitrogen. Sets of experiments were performed so as to promote cell growth in the first stage (supplied with initial xylose 15 g/L), followed by an accumulation phase, where N or P was the limiting nutrient when xylose was fed in pulses to avoid concentrations lower than 5 g/L. N-limited fed-batch specific cell growth (around 0.19 1/h) and substrate consumption (around 0.24 1/h) rates were higher when compared to phosphorus-limited ones. Xylose to PHA yield was similar in both conditions [0.37 gP(3HB)/gxyl]. We also described pst gene cluster in B. sacchari, responsible for high-affinity phosphate uptake. Obtained phosphorus to biomass yields might evidence polyphosphate accumulation. Results were compared with studies with B. sacchari and other PHA-producing microorganisms. Since it is the first report of the mentioned kinetic parameters for LMG 19450 growing on xylose solely, our results open exciting perspectives to develop an efficient bioprocess strategy with increased P(3HB) production from xylose or xylose-rich substrates.

Engineering xylose metabolism for production of polyhydroxybutyrate in the non-model bacterium Burkholderia sacchari.

BACKGROUND: Despite its ability to grow and produce high-value molecules using renewable carbon sources, two main factors must be improved to use Burkholderia sacchari as a chassis for bioproduction at an industrial scale: first, the lack of molecular tools to engineer this organism and second, the inherently slow growth rate and poly-3-hydroxybutyrate [P(3HB)] production using xylose. In this work, we have addressed both factors. RESULTS: First, we adapted a set of BglBrick plasmids and showed tunable expression in B. sacchari. Finally, we assessed growth rate and P(3HB) production through overexpression of xylose transporters, catabolic or regulatory genes. Overexpression of xylR significantly improved growth rate (55.5% improvement), polymer yield (77.27% improvement), and resulted in 71% of cell dry weight as P(3HB). CONCLUSIONS: These values are unprecedented for P(3HB) accumulation using xylose as a sole carbon source and highlight the importance of precise expression control for improving utilization of hemicellulosic sugars in B. sacchari.

A non-naturally-occurring P(3HB-co-3HAMCL) is produced by recombinant Pseudomonas sp. from an unrelated carbon source.

Pseudomonas sp. PHA- was used as host for PHA biosynthesis genes from Aeromonas sp. to produce 3HB-co-3HAMCL from glucose with no supply of co-substrates. A non-naturally-occurring PHA composed mainly of 3HB, 3HHx and 3HD (3HO, 3HDdΔ5 and 3HDd monomers were detected in smaller amounts) was obtained. The polymer was extracted using two different solvents (acetone and chloroform) and subject to the following characterization tests: FTIR, DSC, TGA and GPC. The latter suggests a block copolymer since a single and narrow elution peak was observed for each sample. The DSC results ruled out the possibility of a random copolymer and agrees with a single copolymer composed of two blocks: one with the typical composition of PHAMCL produced by Pseudomonas and another containing 3HB and 3HHx with a high 3HHx molar fraction. Thus, this study increases the perspectives of P(3HB-co-3HAMCL) production from carbohydrates as the sole carbon source.

xylA and xylB overexpression as a successful strategy for improving xylose utilization and poly-3-hydroxybutyrate production in Burkholderia sacchari.

Despite the versatility and many advantages of polyhydroxyalkanoates as petroleum-based plastic substitutes, their higher production cost compared to petroleum-based polymers has historically limited their large-scale production. One appealing approach to reducing production costs is to employ less expensive, renewable feedstocks. Xylose, for example is an abundant and inexpensive carbon source derived from hemicellulosic residues abundant in agro-industrial waste (sugarcane bagasse hemicellulosic hydrolysates). In this work, the production of poly-3-hydroxybutyrate P(3HB) from xylose was studied to develop technologies for conversion of agro-industrial waste into high-value chemicals and biopolymers. Specifically, this work elucidates the organization of the xylose assimilation operon of Burkholderia sacchari, a non-model bacterium with high capacity for P(3HB) accumulation. Overexpression of endogenous xylose isomerase and xylulokinase genes was successfully assessed, improving both specific growth rate and P(3HB) production. Compared to control strain (harboring pBBR1MCS-2), xylose utilization in the engineered strain was substantially improved with 25% increase in specific growth rate, 34% increase in P(3HB) production, and the highest P(3HB) yield from xylose reported to date for B. sacchari (YP3HB/Xil = 0.35 g/g). This study highlights that xylA and xylB overexpression is an effective strategy to improve xylose utilization and P(3HB) production in B. sacchari.