N2-Fixing Cyanobacteria Harnessed for Biosolar Production of Nitrofertilizer

Shengni Tian, Huilan Zhu, Liping Gu, and Ruanbao Zhou

Department of Biology and Microbiology, South Dakota State University, Brookings, SD, United States

IntroductionIn N2-fixing cyanobacteria such as Anabaena sp., ammonium synthesized by the nitrogenase is

assimilated through the sequential reaction of two enzymes, glutamine synthetase (GS) and

glutamate synthase (GOGAT), in a pathway called GS-GOGAT cycle (Fig. 1B). Normally, the

amount of ammonia produced by the heterocysts of Anabaena is tightly regulated to be just

sufficient to support the growth of vegetative cells, with little or no excess of ammonia to

secrete. However, reducing glutamine synthetase (GlnA) activity in vivo could lead to

ammonia secretion (1-2). We hypothesized that genetically knocking down the GlnA activity

would increase the secreting rate of ammonia produced by the heterocystous nitrogenase,

which may further enhance the capability of nitrogen fixation in heterocyst because of timely

removal of ammonia present in heterocyst, an inhibitor of nitrogenase. Therefore, our first

objective of this project is to create an ammonia secreting Anabaena mutant strain.

Abstract

Results

Both M2083 and M2084 mutant strains secreted significant amount of NH3

Conclusions

AcknowledgementsThis research is supported by USDA-NIFAgrant 11665597 (to RZ) and by the SouthDakota Agricultural Experiment Station.

Nitrogen fertilizer is a primary driver in agricultural productivity, and thus impacts food, feed,fiber, and fuel production. Unfortunately, current fossil fuel-dependent ammonia production isenergy intensive and damaging to the environment. Economic and eco-friendly methods toproduce ammonia are urgently needed. Fortunately, N2-fixation by cyanobacterial heterocystsoffers such a unique opportunity. When combined nitrogen becomes limiting, thecyanobacterium Anabaena species responds by the formation of terminally differentiated,specialized nitrogen-fixing cells called heterocysts (Fig. 1A). However, mankind has largelyignored its potential application for this solar-powered, oxic N2-fixation in heterocysts.This seed project is focused on harnessing heterocysts, the solar-powdered N2-fixing cells, forefficient biosolar production of ammonia. The specific objectives for this seed project are:1. Engineering Anabaena to produce and secrete ammonia;2. Creating a desired mutant with higher frequency of functional heterocysts;3. Genetic shunting Anabaena’s nitrogen flow from producing nitrogen-enriched storagepolymers (cyanophycin) to the production of excreted ammonia.

Construction of glnA knockout mutant (M2083) Figure 2-I. Screen for the potential glnA knockoutmutants. A single crossover approach was appliedto knockout glnA by constructing the cargoplasmid pZR2083 containing an internal fragmentof glnA (A). The Anabaena culture mated with E.coli bearing pZR2083 was spread onto NCmembrane atop AA (N) agar containing SP10 for 0(B) and 15 days (C). As a negative control, theconjugal transfer mixture without cargo plasmidshowed no colonies (data not shown) grown onAA (N) agar plate containing SP10 (10 µg/mlspectinomycin).

Conjugaltransfer

pZR2083

Conjugaltransfer

Construction of glnA knockdown mutant (M2084) in which an antisense glnA gene is expressed

Figure 2-II. Screen for the potential glnA knock-down mutants.Expressing antisense approach was applied to knock-down glnA byconstructing the replicable plasmid pZR2084 containingcomplimentary DNA sequence of glnA (left panel). The Anabaenaculture mated with E. coli bearing pZR2084 was spread onto NCmembrane atop AA (N) agar containing Em10 for 15 days (rightpanel). As a negative control, the conjugal transfer mixture withoutpZR2084 showed no colonies (data not shown) grown on AA (N)agar plate containing Em10 (10 µg/ml Erythromycin).

WT M2083-1 M2083-2 M2083-3 WT M2084-3 M2084-7 M2084-9

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Ammonia Secretion after Combined Nitrogen Depletion

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Fig. 1. Heterocyst formation in Anabaena species (A) and ammonia secretion mechanism

Fig. 3. Colony PCR verification of M2083 (A) and M2084 (B)

Fig. 4. M2083 and M2084 can grow well in AA/8 medium (without free combined nitrogen)

Fig. 5. Ammonia production and secretion by M2083, M2084 using N2 gas and light energyXXh-N: deplete free combined nitrogen for xx hours; ammonia secretion were the average of three biological repeats. 15d+N: culture grown with nitrate

M2083 secreted 5, 3, 20-fold more ammonia than the wild-type strain at 26, 48, and 72 hours, respectively M2084 secreted 108, 16, 13-fold more ammonia than the wild-type strain at 26, 48, and 72 hours, respectively

Two Anabaena mutants having glutamine synthetase gene (glnA) knock-down were created. These two glnA knock-down mutants secret up to 108-fold more ammonia than the wild-type.

References1. Healy FG1, Latorre C, Albrecht SL, Reddy PM, Shanmugam KT. 2003. Altered kinetic properties of tyrosine-183 to cysteine mutation inglutamine synthetase of anabaena variabilis strain SA1 is responsible for excretion of ammonium ion produced by nitrogenase. Curr Microbiol.46(6):423-31.2. Qin JD, N Shao, DJ Shi, XD Xu, JD Zhang, PZ Guo, WQ Wang, PS Tang. 1999. Establishment of a highly efficient ammonia secreting mutant ofSynechococcus sp. PCC7942 and the glutamine synthetase activity, photosynthesis and growth in its immobilized cells. Acta Botanica Sinica.41(1):65-70.

WT C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9

(B) Colony PCR verification of M2084(Anabaena sp. PCC7120 bearingpZR2084) using specific primers ZR1368and ZR1369 shown that M2084 strains(C-3, C-7, and C-9) did contain pZR2084replicable plasmid.

(A) Colony PCR verification of M2083 (Anabaena::pZR2083) using specific primers ZR1368A and ZR90(lanes 1-4) shown that pZR2083 did integrate intoAnabaena chromosome; PCR with primers ZR1368A andZR1544 (lanes 5-8) amplified glnA sequence, indicatingthat M2083 has not completely segregated yet.

C-1 C-2 C-3 WT C-1 C-2 C-3 WT

1 2 3 4 5 6 7 8

A BKb

1.71.31.10.70.5

Both the verified M2083 and M2084 are capable of growing nearly as well

as the wild-type using atmospheric N2 gas as the sole nitrogen source

Genetic Verification of glnA mutants M2083 and M2084 by Colony PCR

Creating Two GlnA Mutants M2083 and M2084 for Ammonia Secretion

A B C